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Cisplatin is a widely used anti-cancer drug used to treat a variety of cancer types. One of the side effects of this life-saving drug is irreversible ototoxicity, resulting in permanent hearing loss in many patients. In order to understand why cisplatin is particularly toxic to the inner ear, we compared the hearing loss and cochlear uptake of cisplatin to that of two related drugs, carboplatin and oxaliplatin. These three drugs are similar in that each contains a core platinum atom; however, carboplatin and oxaliplatin are considered less ototoxic than cisplatin. We delivered these three drugs to mice using a 6-week cyclic drug administration protocol. We performed the experiment twice, once using equimolar concentrations of the drugs and once using concentrations of the drugs more proportional to those used in the clinic. For both concentrations, we detected a significant hearing loss caused by cisplatin and no hearing loss caused by carboplatin or oxaliplatin. Cochlear uptake of each drug was measured using inductively coupled plasma mass spectrometry (ICP-MS) to detect platinum. Cochlear platinum levels were highest in mice treated with cisplatin followed by oxaliplatin, while carboplatin was largely excluded from the cochlea. Even when the drug doses were increased, cochlear platinum remained low in mice treated with oxaliplatin or carboplatin. We also examined drug clearance from the inner ear by measuring platinum levels at 1 h and 24 h after drug administration. Our findings suggest that the reduced cochlear platinum we observed with oxaliplatin and carboplatin were not due to increased clearance of these drugs relative to cisplatin. Taken together, our data indicate that the differential ototoxicity among cisplatin, carboplatin, and oxaliplatin is attributable to differences in cochlear uptake of these three drugs.

Hair cells, the mechanosensory receptors of the inner ear, are responsible for hearing and balance. Hair cell death and consequent hearing loss are common results of treatment with ototoxic drugs, including the widely used aminoglycoside antibiotics. Induction of heat shock proteins (HSPs) confers protection against aminoglycoside-induced hair cell death via paracrine signaling that requires extracellular heat shock 70-kDa protein (HSP70). We investigated the mechanisms underlying this non–cell-autonomous protective signaling in the inner ear. In response to heat stress, inner ear tissue releases exosomes that carry HSP70 in addition to canonical exosome markers and other proteins. Isolated exosomes from heat-shocked utricles were sufficient to improve survival of hair cells exposed to the aminoglycoside antibiotic neomycin, whereas inhibition or depletion of exosomes from the extracellular environment abolished the protective effect of heat shock. Hair cell–specific expression of the known HSP70 receptor TLR4 was required for the protective effect of exosomes, and exosomal HSP70 interacted with TLR4 on hair cells. Our results indicate that exosomes are a previously undescribed mechanism of intercellular communication in the inner ear that can mediate nonautonomous hair cell survival. Exosomes may hold potential as nanocarriers for delivery of therapeutics against hearing loss.

Dizziness and hearing loss are among the most common disabilities. Many forms of hereditary balance and hearing disorders are caused by abnormal development of stereocilia, mechanosensory organelles on the apical surface of hair cells in the inner ear. The deaf whirler mouse, a model of human Usher syndrome (manifested by hearing loss, dizziness, and blindness), has a recessive mutation in the whirlin gene, which renders hair cell stereocilia short and dysfunctional. In this study, wild-type whirlin cDNA was delivered to the inner ears of neonatal whirler mice using adeno-associated virus serotype 2/8 (AAV8-whirlin) by injection into the posterior semicircular canal. Unilateral whirlin gene therapy injection was able to restore balance function as well as improve hearing in whirler mice for at least 4 months. Our data indicate that gene therapy is likely to become a treatment option for hereditary disorders of balance and hearing.

The endocochlear potential (EP) generated by the stria vascularis (SV) is necessary for hair cell mechanotransduction in the mammalian cochlea. We sought to create a model of EP dysfunction for the purposes of transcriptional analysis and treatment testing. By administering a single dose of cisplatin, a commonly prescribed cancer treatment drug with ototoxic side effects, to the adult mouse, we acutely disrupt EP generation. By combining these data with single cell RNA-sequencing findings, we identify transcriptional changes induced by cisplatin exposure, and by extension transcriptional changes accompanying EP reduction, in the major cell types of the SV. We use these data to identify gene regulatory networks unique to cisplatin treated SV, as well as the differentially expressed and druggable gene targets within those networks. Our results reconstruct transcriptional responses that occur in gene expression on the cellular level while identifying possible targets for interventions not only in cisplatin ototoxicity but also in EP dysfunction.

The mouse utricle model system is the best-characterized ex vivo preparation for studies of mature mammalian hair cells (HCs). Despite the many advantages of this model system, efficient and reliable quantification of HCs from cultured utricles has been a persistent challenge with this model system. Utricular HCs are commonly quantified by counting immunolabeled HCs in regions of interest (ROIs) placed over an image of the utricle. Our data indicate that the accuracy of HC counts obtained using this method can be impacted by variability in HC density across different regions of the utricle. In addition, the commonly used HC marker myosin 7a results in a diffuse cytoplasmic stain that is not conducive to automated quantification and must be quantified manually, a labor-intensive task. Furthermore, myosin 7a immunoreactivity is retained in dead HCs, resulting in inaccurate quantification of live HCs using this marker. Here we have developed a method for semi-automated quantification of surviving HCs that combines immunoreactivity for the HC-specific transcription factor Pou4f3 with labeling of activated caspase 3/7 (AC3/7) to detect apoptotic HCs. The discrete nuclear Pou4f3 signal allowed us to utilize the binary or threshold function within ImageJ to automate HC quantification. To further streamline this process, we created an ImageJ macro that automates the process from raw image loading to a final quantified image that can be immediately evaluated for accuracy. Within this quantified image, the user can manually correct the quantification via an image overlay indicating the counted HC nuclei. Pou4f3-positive HCs that also express AC3/7 are subtracted to yield accurate counts of surviving HCs. Overall, we present a semi-automated method that is faster than manual HC quantification and identifies surviving HCs with high accuracy.

BACKGROUND: Cisplatin is widely used to treat adult and pediatric cancers. It is the most ototoxic drug in clinical use, resulting in permanent hearing loss in approximately 50% of treated patients. There is a major need for therapies that prevent cisplatin-induced hearing loss. Studies in mice suggest that concurrent use of statins reduces cisplatin-induced hearing loss. METHODS: We examined hearing thresholds from 277 adults treated with cisplatin for head and neck cancer. Pretreatment and posttreatment audiograms were collected within 90 days of initiation and completion of cisplatin therapy. The primary outcome measure was a change in hearing as defined by the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE). RESULTS: Among patients on concurrent atorvastatin, 9.7% experienced a CTCAE grade 2 or higher cisplatin-induced hearing loss compared with 29.4% in nonstatin users (P < 0.0001). A mixed-effect model analysis showed that atorvastatin use was significantly associated with reduced cisplatin-induced hearing loss (P ≤ 0.01). An adjusted odds ratio (OR) analysis indicated that an atorvastatin user is 53% less likely to acquire a cisplatin-induced hearing loss than a nonstatin user (OR = 0.47; 95% CI, 0.30–0.78). Three-year survival rates were not different between atorvastatin users and nonstatin users (P > 0.05). CONCLUSIONS: Our data indicate that atorvastatin use is associated with reduced incidence and severity of cisplatin-induced hearing loss in adults being treated for head and neck cancer. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT03225157. FUNDING: Funding was provided by the Division of Intramural Research at the National Institute on Deafness and Other Communication Disorders (1 ZIA DC000079, ZIA DC000090).

Cisplatin-induced ototoxicity results in significant, permanent hearing loss in pediatric and adult cancer survivors. Elucidating the mechanisms underlying cisplatin-induced hearing loss as well as the development of therapies to reduce and/or reverse cisplatin ototoxicity have been impeded by suboptimal animal models. Clinically, cisplatin is most commonly administered in multi-dose, multi-cycle protocols. However, many animal studies are conducted using single injections of high-dose cisplatin, which is not reflective of clinical cisplatin administration protocols. Significant limitations of both high-dose, single-injection protocols and previous multi-dose protocols in rodent models include high mortality rates and relatively small changes in hearing sensitivity. These limitations restrict assessment of both long-term changes in hearing sensitivity and effects of potential protective therapies. Here, we present a detailed method for an optimized mouse model of cisplatin ototoxicity that utilizes a multi-cycle administration protocol that better approximates the type and degree of hearing loss observed clinically. This protocol results in significant hearing loss with very low mortality. This mouse model of cisplatin ototoxicity provides a platform for examining mechanisms of cisplatin-induced hearing loss as well as developing therapies to protect the hearing of cancer patients receiving cisplatin therapy.

Hereditary deafness is one of the most common disabilities affecting newborns. Many forms of hereditary deafness are caused by morphological defects of the stereocilia bundles on the apical surfaces of inner ear hair cells, which are responsible for sound detection. We explored the effectiveness of gene therapy in restoring the hair cell stereocilia architecture in the whirlin mouse model of human deafness, which is deaf due to dysmorphic, short stereocilia. Wild-type whirlin cDNA was delivered via adeno-associated virus (AAV8) by injection through the round window of the cochleas in neonatal whirler mice. Subsequently, whirlin expression was detected in infected hair cells (IHCs), and normal stereocilia length and bundle architecture were restored. Whirlin gene therapy also increased inner hair cell survival in the treated ears compared to the contralateral nontreated ears. These results indicate that a form of inherited deafness due to structural defects in cochlear hair cells is amenable to restoration through gene therapy.

Cisplatin is used to treat a variety of solid tumors in both children and adults. However, cisplatin has serious side-effects, some of which may permanently affect patients' quality of life following treatment, such as ototoxicity. There is currently no FDA-approved therapy for the prevention or treatment of cisplatin-induced hearing loss. Herein we examine the potential for statins to prevent cisplatin-induced ototoxicity. Statins, a class of drugs commonly used to prevent or manage hypercholesterolemia, have been of clinical utility for decades with dependable outcomes and reliable safety profiles in humans. Statins are known to be protective in animal models of noise-induced and age-related hearing loss. Moreover, studies have demonstrated an additive benefit of statins in cancer treatment. In the current study, lovastatin reduces cisplatin-induced hearing loss in adult mice. Lovastatin-mediated protection was significantly greater among female than male mice, and the dose of lovastatin required for protection was different between the sexes. Taken together our data indicate that lovastatin reduces cisplatin-induced hearing loss in mice and suggest that concurrent statin and cisplatin therapy may represent a feasible clinical strategy for reducing cisplatin-induced ototoxicity that should be explored for future clinical use.

Immune checkpoint inhibition used in combination with standard cisplatin-based chemotherapy regimens is currently under evaluation in clinical trials for head and neck squamous cell carcinoma (HNSCC). The impact of anti–PD-1 therapy on cisplatin-induced ototoxicity and nephrotoxicity has not been established. Here we use a murine model of cisplatin-induced hearing loss to investigate the impact of anti–PD-1 immunotherapy on auditory brainstem responses (ABRs), distortion product otoacoustic emissions (DPOAEs), serum creatinine, and hair cell and renal histology. We demonstrate only mild worsening of DPOAEs at 14.4 and 16 kHz as well as a mild increase in serum creatinine. Renal and hair cell histology as well as ABR measures were unchanged by PD-1 inhibition. Thus, our data suggest that the use of PD-1 inhibition in conjunction with cisplatin results in toxicities that are similar to those of cisplatin alone.

Objectives/Hypothesis Gene therapy offers the possibility of delivering corrective genetic materials to the cochlea, potentially improving hearing. In animals, the most commonly used surgical methods for viral gene therapy delivery to the cochlea are the round window and the cochleostomy approaches. However, the patterns of viral infection and the effects on hearing have not been directly compared between these two approaches. In this study, we compare the patterns of cochlear infection and effects on hearing between these two surgical approaches using adeno-associated virus serotype 2/8 (AAV8) as the gene delivery vehicle. Study Design Animal study and basic science research. Methods One- to two-month-old CBA/J mice were used in this study. AAV8-green fluorescent protein (GFP) was delivered to the cochlea by either the round window or the cochleostomy approach (described below). Auditory brainstem response was used to examine hearing thresholds before and after surgery. Animals were examined at 1, 2, 3, and 4 weeks after surgery for the patterns of cochlear infection and hearing loss. Results Cochlear gene transfer was successful through both surgical approaches. In both approaches, AAV8-GFP mostly infected the inner hair cells. There was occasional low-level infection of the outer hair cells and supporting cells. The two surgical approaches resulted in comparable viral infection efficiencies. The round window approach resulted in less surgical trauma, as indicated by hearing loss, than the cochleostomy approach. Conclusions Adeno-associated virus-mediated gene transfer to the cochlea can be accomplished using either the round window or the cochleostomy surgical approach. The round window approach resulted in less hearing loss compared to the cochleostomy approach. Level of Evidence NA Laryngoscope, 125:2557–2564, 2015

Gene therapy is a promising treatment modality that is being explored for several inherited disorders. Multiple human gene therapy clinical trials are currently ongoing, but few are directed at hearing loss. Hearing loss is one of the most prevalent sensory disabilities in the world, and genetics play an important role in the pathophysiology of hearing loss. Gene therapy offers the possibility of restoring hearing by overcoming the functional deficits created by the underlying genetic mutations. In addition, gene therapy could potentially be used to induce hair cell regeneration by delivering genes that are critical to hair cell differentiation into the cochlea. In this review, we examine the promises and challenges of applying gene therapy to the cochlea. We also summarize recent studies that have applied gene therapy to animal models of hearing loss.

Cisplatin chemotherapy causes permanent hearing loss in 40–80% of treated patients. It is unclear whether the cochlea has unique sensitivity to cisplatin or is exposed to higher levels of the drug. Here we use inductively coupled plasma mass spectrometry (ICP-MS) to examine cisplatin pharmacokinetics in the cochleae of mice and humans. In most organs cisplatin is detected within one hour after injection, and is eliminated over the following days to weeks. In contrast, the cochlea retains cisplatin for months to years after treatment in both mice and humans. Using laser ablation coupled to ICP-MS, we map cisplatin distribution within the human cochlea. Cisplatin accumulation is consistently high in the stria vascularis, the region of the cochlea that maintains the ionic composition of endolymph. Our results demonstrate long-term retention of cisplatin in the human cochlea, and they point to the stria vascularis as an important therapeutic target for preventing cisplatin ototoxicity., Permanent hearing loss occurs in many cancer patients treated with cisplatin. In this study, the authors examine cisplatin pharmacokinetics in the cochleae of mice and humans showing that cisplatin is retained for months to years after treatment.

The first major recognition of drug-induced hearing loss can be traced back more than seven decades to the development of streptomycin as an antimicrobial agent. Since then at least 130 therapeutic drugs have been recognized as having ototoxic side- effects. Two important classes of ototoxic drugs are the aminoglycoside antibiotics and the antineoplastic agent cisplatin. These drugs save the lives of millions of people worldwide, but they also cause irreparable hearing loss. In the inner ear, sensory hair cells (HCs) and spiral ganglion neurons (SGNs) are important cellular targets of these drugs, and most mechanistic studies have focused on the cell-autonomous responses of these cell types in response to ototoxic stress. Despite several decades of studies on ototoxicity, important unanswered questions remain, including the cellular and molecular mechanisms that determine whether HCs and SGNs will live or die when confronted with ototoxic challenge. Emerging evidence indicates that other cell types in the inner ear can act as mediators of survival or death of sensory cells and spiral ganglion neurons. For example, glia-like supporting cells (SCs) can promote survival of both HCs and SGNs. Alternatively, SCs can act to promote HC death and inhibit neural fiber expansion. Similarly, tissue resident macrophages activate either pro-survival or pro-death signaling that can influence HC survival after exposure to ototoxic agents. Together these data indicate that autonomous responses that occur within a stressed HC or SGN are not the only (and possibly not the primary) determinants of whether the stressed cell ultimately lives or dies. Instead non-autonomous cellular responses are emerging as significant determinants of HC and SGN survival vs. death in the face of ototoxic stress. The goal of this review is to summarize the current evidence on non-autonomous cellular responses to ototoxic stress and to discuss ways in which this knowledge may advance the development of therapies to reduce hearing loss caused by these drugs.

Mechanosensory hair cells are the receptor cells of hearing and balance. Hair cells are sensitive to death from exposure to therapeutic drugs with ototoxic side effects, including aminoglycoside antibiotics and cisplatin. We recently showed that the induction of heat shock protein 70 (HSP70) inhibits ototoxic drug–induced hair cell death. Here, we examined the mechanisms underlying the protective effect of HSP70. In response to heat shock, HSP70 was induced in glia-like supporting cells but not in hair cells. Adenovirus-mediated infection of supporting cells with Hsp70 inhibited hair cell death. Coculture with heat-shocked utricles protected nonheat-shocked utricles against hair cell death. When heat-shocked utricles from Hsp70–/– mice were used in cocultures, protection was abolished in both the heat-shocked utricles and the nonheat-shocked utricles. HSP70 was detected by ELISA in the media surrounding heat-shocked utricles, and depletion of HSP70 from the media abolished the protective effect of heat shock, suggesting that HSP70 is secreted by supporting cells. Together our data indicate that supporting cells mediate the protective effect of HSP70 against hair cell death, and they suggest a major role for supporting cells in determining the fate of hair cells exposed to stress.

Background: Delivery of therapeutic agents directly through the round window (RW) offers promise for treating sensorineural hearing loss. However, hearing loss can result from the surgical approach itself, and the reasons for this are poorly understood. We examined the hearing loss following the 3 major steps involved with the RW approach to access the mouse cochlea: bullostomy, RW puncture, and RW injection. Methods: Twenty-one adult CBA/J mice underwent bullostomy alone, 10 underwent RW puncture, and 8 underwent RW injection with PBS with 5% glycerol. Auditory brainstem responses (ABR) and otoscopy were performed preoperatively and up to 6 weeks postoperatively. Hair cells were stained, and survival was assessed using immunofluorescence. Results: One week postoperatively, mice in all groups showed significant threshold shifts. Otoscopy revealed approximately half of all mice had middle ear effusion (MEE), with a higher incidence of effusion in the RW puncture and RW injection groups. Those with MEE had significant ABR threshold shifts, whereas those without MEE had minimal hearing loss. MEE persisted through 6 weeks in a majority of cases, but in those mice with MEE resolution, there was at least partial improvement in hearing. Immunohistochemistry showed minimal loss of hair cells in all animals. Conclusion: MEE is highly correlated with hearing loss in mice undergoing RW surgery. Otoscopy is an important adjunct to consider after ear surgery in mice, as MEE may contribute to postsurgical hearing loss.

The hair cells of the larval zebrafish lateral line provide a useful preparation in which to study hair cell death and to screen for genes and small molecules that modulate hair cell toxicity. We recently reported preliminary results from screening a small-molecule library for compounds that inhibit aminoglycoside-induced hair cell death. To potentially reduce the time required for development of drugs and drug combinations that can be clinically useful, we screened a library of 1,040 FDA-approved drugs and bioactive compounds (NINDS Custom Collection II). Seven compounds that protect against neomycin-induced hair cell death were identified. Four of the seven drugs inhibited aminoglycoside uptake, based on Texas-Red-conjugated gentamicin uptake. The activities of two of the remaining three drugs were evaluated using an in vitro adult mouse utricle preparation. One drug, 9-amino-1,2,3,4-tetrahydroacridine (tacrine) demonstrated conserved protective effects in the mouse utricle. These results demonstrate that the zebrafish lateral line can be used to screen successfully for drugs within a library of FDA-approved drugs and bioactives that inhibit hair cell death in the mammalian inner ear and identify tacrine as a promising protective drug for future studies.

Sensory hair cells of the inner ear are sensitive to death from aging, noise trauma, and ototoxic drugs. Ototoxic drugs include the aminoglycoside antibiotics and the antineoplastic agent cisplatin. Exposure to aminoglycosides results in hair cell death that is mediated by specific apoptotic proteins, including c-Jun N-terminal kinase (JNK) and caspases. Induction of heat shock proteins (Hsps) is a highly conserved stress response that can inhibit JNK- and caspase-dependent apoptosis in a variety of systems. We have previously shown that heat shock results in a robust upregulation of Hsps in the hair cells of the adult mouse utricle in vitro. In addition, heat shock results in significant inhibition of both cisplatin- and aminoglycoside-induced hair cell death. In our system, Hsp70 is the most strongly induced Hsp, which is upregulated over 250-fold at the level of mRNA 2 h after heat shock. Therefore, we have begun to examine the role of Hsp70 in mediating the protective effect of heat shock. To determine whether Hsp70 is necessary for the protective effect of heat shock against aminoglycoside-induced hair cell death, we utilized utricles from Hsp70.1/3 (-/-) mice. While heat shock inhibited gentamicin-induced hair cell death in wild-type utricles, utricles from Hsp70.1/3 (-/-) mice were not protected. In addition, we have examined the role of the major heat shock transcription factor, Hsf1, in mediating the protective effect of heat shock. Utricles from Hsf1 (-/-) mice and wild-type littermates were exposed to heat shock followed by gentamicin. The protective effect of heat shock on aminoglycoside-induced hair cell death was only observed in wild-type mice and not in Hsf1 (-/-) mice. To determine whether Hsp70 is sufficient to protect hair cells, we have utilized transgenic mice that constitutively overexpress Hsp70. Utricles from Hsp70-overexpressing mice and wild-type littermates were cultured in the presence of varying neomycin concentrations for 24 h. The Hsp70-overexpressing utricles were significantly protected against neomycin-induced hair cell death at moderate to high doses of neomycin. This protective effect was achieved without a heat shock. Taken together, these data indicate that Hsp70 and Hsf1 are each necessary for the protective effect of heat shock against aminoglycoside-induced death. Furthermore, overexpression of Hsp70 alone significantly inhibits aminoglycoside-induced hair cell death.

The zebrafish is a valuable model for studying hair cell development, structure, genetics, and behavior. Zebrafish and other aquatic vertebrates have hair cells on their body surface organized into a sensory system called the lateral line. These hair cells are highly accessible and easily visualized using fluorescent dyes. Morphological and functional similarities to mammalian hair cells of the inner ear make the zebrafish a powerful preparation for studying hair cell toxicity. The ototoxic potential of drugs has historically been uncovered by anecdotal reports that have led to more formal investigation. Currently, no standard screen for ototoxicity exists in drug development. Thus, for the vast majority of Food and Drug Association (FDA)-approved drugs, the ototoxic potential remains unknown. In this study, we used 5-day-old zebrafish larvae to screen a library of 1,040 FDA-approved drugs and bioactives (NINDS Custom Collection II) for ototoxic effects in hair cells of the lateral line. Hair cell nuclei were selectively labeled using a fluorescent vital dye. For the initial screen, fish were exposed to drugs from the library at a 100-muM concentration for 1 h in 96-well tissue culture plates. Hair cell viability was assessed in vivo using fluorescence microscopy. One thousand forty drugs were rapidly screened for ototoxic effects. Seven known ototoxic drugs included in the library, including neomycin and cisplatin, were positively identified using these methods, as proof of concept. Fourteen compounds without previously known ototoxicity were discovered to be selectively toxic to hair cells. Dose-response curves for all 21 ototoxic compounds were determined by quantifying hair cell survival as a function of drug concentration. Dose-response relationships in the mammalian inner ear for two of the compounds without known ototoxicity, pentamidine isethionate and propantheline bromide, were then examined using in vitro preparations of the adult mouse utricle. Significant dose-dependent hair cell loss in the mouse utricle was demonstrated for both compounds. This study represents an important step in validating the use of the zebrafish lateral line as a screening tool for the identification of potentially ototoxic drugs.

The maintenance of sensory hair cell stereocilia is critical for lifelong hearing; however, mechanisms of structural homeostasis remain poorly understood. Conflicting models propose that stereocilia F-actin cores are either continually renewed every 24–48 h via a treadmill or are stable, exceptionally long-lived structures. Here to distinguish between these models, we perform an unbiased survey of stereocilia actin dynamics in more than 500 utricle hair cells. Live-imaging EGFP-β-actin or dendra2-β-actin reveal stable F-actin cores with turnover and elongation restricted to stereocilia tips. Fixed-cell microscopy of wild-type and mutant β-actin demonstrates that incorporation of actin monomers into filaments is required for localization to stereocilia tips. Multi-isotope imaging mass spectrometry and live imaging of single differentiating hair cells capture stereociliogenesis and explain uniform incorporation of 15N-labelled protein and EGFP-β-actin into nascent stereocilia. Collectively, our analyses support a model in which stereocilia actin cores are stable structures that incorporate new F-actin only at the distal tips., Precise control of stereocilia length by auditory hair cells is vital for normal hearing. Drummond et al. follow in real-time the incorporation of actin into these structures and show that while the actin core is remarkably stable, and actin polymerization is limited to their distal tips.

Recruitment of endogenous progenitors is critical during tissue repair. The inner ear utricle requires mechanosensory hair cells (HCs) to detect linear acceleration. After damage, non-mammalian utricles regenerate HCs via both proliferation and direct transdifferentiation. In adult mammals, limited transdifferentiation from unidentified progenitors occurs to regenerate extrastriolar Type II HCs. Here, we show that HC damage in neonatal mouse utricle activates the Wnt target gene Lgr5 in striolar supporting cells. Lineage tracing and time-lapse microscopy reveal that Lgr5+ cells transdifferentiate into HC-like cells in vitro. In contrast to adults, HC ablation in neonatal utricles in vivo recruits Lgr5+ cells to regenerate striolar HCs through mitotic and transdifferentiation pathways. Both Type I and II HCs are regenerated, and regenerated HCs display stereocilia and synapses. Lastly, stabilized β-catenin in Lgr5+ cells enhances mitotic activity and HC regeneration. Thus Lgr5 marks Wnt-regulated, damage-activated HC progenitors and may help uncover factors driving mammalian HC regeneration.

Gene therapy offers the possibility of delivering corrective genetic materials to the cochlea, potentially improving hearing. In animals, the most commonly used surgical methods for viral gene therapy delivery to the cochlea are the round window and the cochleostomy approaches. However, the patterns of viral infection and the effects on hearing have not been directly compared between these two approaches. In this study, we compare the patterns of cochlear infection and effects on hearing between these two surgical approaches using adeno-associated virus serotype 2/8 (AAV8) as the gene delivery vehicle.Animal study and basic science research.One- to two-month-old CBA/J mice were used in this study. AAV8-green fluorescent protein (GFP) was delivered to the cochlea by either the round window or the cochleostomy approach (described below). Auditory brainstem response was used to examine hearing thresholds before and after surgery. Animals were examined at 1, 2, 3, and 4 weeks after surgery for the patterns of cochlear infection and hearing loss.Cochlear gene transfer was successful through both surgical approaches. In both approaches, AAV8-GFP mostly infected the inner hair cells. There was occasional low-level infection of the outer hair cells and supporting cells. The two surgical approaches resulted in comparable viral infection efficiencies. The round window approach resulted in less surgical trauma, as indicated by hearing loss, than the cochleostomy approach.Adeno-associated virus-mediated gene transfer to the cochlea can be accomplished using either the round window or the cochleostomy surgical approach. The round window approach resulted in less hearing loss compared to the cochleostomy approach.NA.

Mechanosensory hair cells are susceptible to apoptotic death in response to exposure to ototoxic drugs, including aminoglycoside antibiotics. The c-Jun n-terminal kinase (JNK) is a stress-activated protein kinase that can promote apoptotic cell death in a variety of systems. Inhibition of the JNK signaling pathway can prevent aminoglycoside-induced death of cochlear and vestibular sensory hair cells. We used an in vitro preparation of utricles from adult mice to examine the role of JNK activation in aminoglycoside-induced hair cell death. CEP-11004 was used as an indirect inhibitor of JNK signaling. Immunohistochemistry showed that both JNK and its downstream target c-Jun are phosphorylated in hair cells of utricles exposed to neomycin. CEP-11004 inhibited neomycin-induced phosphorylation of both JNK and c-Jun. CEP-11004 inhibited hair cell death in utricles exposed to moderate doses of neomycin. However, the results were not uniform across the dose-response function; CEP-11004 did not inhibit hair cell death in utricles exposed to high-dose neomycin. The CEP-11004-induced protective effect was not due to inhibition of PKC or p38, since neither chelerythrine nor SB203580 could mimic the protective effect of CEP-11004. In addition, inhibition of JNK inhibited the activation of caspase-9 in hair cells. These results indicate that JNK plays an important role in neomycin-induced vestibular hair cell death and caspase-9 activation.

Human hearing and balance impairments are often attributable to the death of sensory hair cells in the inner ear. These cells are hypersensitive to death induced by noise exposure, aging, and some therapeutic drugs. Two major classes of ototoxic drugs are the aminoglycoside antibiotics and the antineoplastic agent cisplatin. Exposure to these drugs leads to hair cell death that is mediated by the activation of specific apoptotic proteins, including caspases. The induction of heat shock proteins (HSPs) in response to cellular stress is a ubiquitous and highly conserved response that can significantly inhibit apoptosis in some systems by inhibiting apoptotic proteins. Induction of HSPs occurs in hair cells in response to a variety of stimuli. Given that HSPs can directly inhibit apoptosis, we hypothesized that heat shock may inhibit apoptosis in hair cells exposed to ototoxic drugs. To test this hypothesis, we developed a method for inducing HSP expression in the adult mouse utricle in vitro. In vitro heat shock reliably produces a robust up-regulation of HSP-70 mRNA and protein, as well as more modest up-regulation of HSP-90 and HSP-27. The heat shock does not result in death of hair cells. Heat shock has a significant protective effect against both aminoglycoside- and cisplatin-induced hair cell death in the utricle preparation in vitro. These data indicate that heat shock can inhibit ototoxic drug-induced hair cell death, and that the utricle preparation can be used to examine the molecular mechanism(s) underlying this protective effect.

Sensory hair cells of the inner ear are susceptible to death from a variety of stresses including aging, noise trauma, genetic disorders, and exposure to certain therapeutic drugs. Ototoxic drugs include the aminoglycoside antibiotics and the antineoplastic agent cisplatin. This is a short technical report describing the dissection and culture of the adult mouse utricle. This in vitro preparation allows for detailed studies of ototoxic-drug-induced hair cell death in an adult mammalian system. In addition, this preparation allows for examination of the effects of specific gene products through the use of transgenic and knockout mouse models.

Sensory hair cells are mechanotransducers of the inner ear that are essential for hearing and balance. Hair cell death commonly occurs following acoustic trauma or exposure to ototoxins, such as the aminoglycoside antibiotics and the antineoplastic agent cisplatin. Loss of these inner ear sensory cells can lead to permanent sensorineural hearing loss, balance disturbance, or both. Currently, the only effective clinical intervention is prevention from exposure to known ototoxic insults. To help improve therapeutic strategies, a better understanding of the molecular mechanisms underlying hair cell degeneration is required. Current knowledge of these cell death mechanisms and potential therapeutic targets are discussed in this review.Studies have shown that caspase-9 and caspase-3 are key mediators of hair cell death induced by noise, aminoglycosides, and cisplatin. The Bcl-2 family consists of a group of proapoptotic and antiapoptotic molecules that act upstream of and regulate caspase activation. Recent studies have shed light on the roles of molecules acting more upstream, including mitogen-activated protein kinases and p53.The mechanisms of sensory hair cell degeneration in response to different ototoxic stimuli share a final common pathway: caspase activation. Inhibition of caspases prevents or delays hair cell death and may preserve hearing/balance function. Inhibition of mitogen-activated protein kinases protects against noise-induced and aminoglycoside-induced but not cisplatin-induced hair cell death, which suggests divergent upstream regulatory mechanisms.

Mechanosensory hair cells of the inner ear are especially sensitive to death induced by exposure to aminoglycoside antibiotics. This aminoglycoside-induced hair cell death involves activation of an intrinsic program of cellular suicide. Aminoglycoside-induced hair cell death can be prevented by broad-spectrum inhibition of caspases, a family of proteases that mediate apoptotic and programmed cell death in a wide variety of systems. More specifically, aminoglycoside-induced hair cell death requires activation of caspase-9. Caspase-9 activation requires release of mitochondrial cytochrome c into the cytoplasm, indicating that aminoglycoside-induced hair cell death is mediated by the mitochondrial (or “intrinsic”) cell death pathway. The Bcl-2 family of pro-apoptotic and anti-apoptotic proteins are important upstream regulators of the mitochondrial apoptotic pathway. Bcl-2 is an anti-apoptotic protein that localizes to the mitochondria and promotes cell survival by preventing cytochrome c release. Here we have utilized transgenic mice that overexpress Bcl-2 to examine the role of Bcl-2 in neomycin-induced hair cell death. Overexpression of Bcl-2 significantly increased hair cell survival following neomycin exposure in organotypic cultures of the adult mouse utricle. Furthermore, Bcl-2 overexpression prevented neomycin-induced activation of caspase-9 in hair cells. These results suggest that the expression level of Bcl-2 has important effects on the pathway(s) important for the regulation of aminoglycoside-induced hair cell death. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 89–100, 2004

Mechanosensory hair cells of the inner ear are susceptible to death when exposed to a variety of drugs including aminoglycoside antibiotics. During avian and mammalian development, there is a period of relative insensitivity to aminoglycoside-induced hair cell death. This study was designed to test the hypothesis that zebrafish (Danio rerio) have developmental differences in sensitivity to aminoglycoside-induced hair cell death in the lateral line neuromasts. Larval zebrafish of various ages were exposed to several concentrations of neomycin, and their hair cells were examined using the potentiometric vital dye, DASPEI. Results indicate that zebrafish larvae aged 4 days post-fertilization are relatively insensitive to aminoglycoside-induced hair cell death compared to older fish. Thus zebrafish hair cells show developmental differences in sensitivity to aminoglycoside-induced death similar to those reported for inner ear hair cells of birds and mammals.

Caspases are a family of proteases that have been implicated as key mediators of cell death. Although nonspecific inhibition of caspase activation has been reported to prevent mammalian sensory hair cell death, the exact roles of individual caspases during hair cell death are unclear. In other systems, the activation of initiator caspases, such as caspase-8 and caspase-9, can lead to the activation of the effector caspase-3. We have begun to systematically characterize hair cell death in an in vitro system by examining the activation of these specific caspases in degenerating hair cells after acutely damaging the whole avian basilar papilla with gentamicin. Basilar papillae (BP) displayed a dose-dependent hair cell loss after a 24-h treatment with gentamicin at concentrations of 0.1, 0.5, and 2.0 mM. When treated with 0.5 mM gentamicin for 6, 12, or 24 h, hair cells first began to degenerate in the basal third of the BP and damage progressed apically. Supplementation of z-VAD-fmk, a general caspase inhibitor, provided short-term protection against gentamicin-induced hair cell death. Treatment with gentamicin for 6 or 12 h promoted the expression of active caspase-3 and active caspase-9 in many hair cells along the BP as shown by immunohistochemistry. At these time-points, specific fluorescent-labeled peptide substrates detected more active caspase-3, caspase-8, and caspase-9 in gentamicin-treated hair cells relative to controls. Our data indicate that auditory hair cells degenerate as a result of gentamicin exposure in a caspase-dependent manner. Specifically, the upstream caspases, caspase-8 and caspase-9, and the downstream caspase-3 are activated in aminoglycoside-damaged hair cells.

Hearing loss and balance disorders affect millions of people worldwide. Sensory transduction in the inner ear requires both mechanosensory hair cells (HCs) and surrounding glia-like supporting cells (SCs). HCs are susceptible to death from aging, noise overexposure, and treatment with therapeutic drugs that have ototoxic side effects; these ototoxic drugs include the aminoglycoside antibiotics and the antineoplastic drug cisplatin. Although both classes of drugs are known to kill HCs, their effects on SCs are less well understood. Recent data indicate that SCs sense and respond to HC stress, and that their responses can influence HC death, survival, and phagocytosis. These responses to HC stress and death are critical to the health of the inner ear. Here we have used live confocal imaging of the adult mouse utricle, to examine the SC responses to HC death caused by aminoglycosides or cisplatin. Our data indicate that when HCs are killed by aminoglycosides, SCs efficiently remove HC corpses from the sensory epithelium in a process that includes constricting the apical portion of the HC after loss of membrane integrity. SCs then form a phagosome, which can completely engulf the remaining HC body, a phenomenon not previously reported in mammals. In contrast, cisplatin treatment results in accumulation of dead HCs in the sensory epithelium, accompanied by an increase in SC death. The surviving SCs constrict fewer HCs and display impaired phagocytosis. These data are supported by in vivo experiments, in which cochlear SCs show reduced capacity for scar formation in cisplatin-treated mice compared with those treated with aminoglycosides. Together, these data point to a broader defect in the ability of the cisplatin-treated SCs, to preserve tissue health in the mature mammalian inner ear.

Prolonged exposure to noise results in loss of ribbon synapses, composed in part by superclusters of neurotransmitter vesicles in the sensory hair cells of the cochlea. Expression of a nerve growth factor in mouse cochlea mitigates noise-induced damage and hearing loss.

Therapeutic drugs with ototoxic side effects cause significant hearing loss for thousands of patients annually. Two major classes of ototoxic drugs are cisplatin and the aminoglycoside antibiotics, both of which are toxic to mechanosensory hair cells, the receptor cells of the inner ear. A critical need exists for therapies that protect the inner ear without inhibiting the therapeutic efficacy of these drugs. The induction of heat shock proteins (HSPs) inhibits both aminoglycoside- and cisplatin-induced hair cell death and hearing loss. We hypothesized that exposure to sound that is titrated to stress the inner ear without causing permanent damage would induce HSPs in the cochlea and inhibit ototoxic drug–induced hearing loss. We developed a sound exposure protocol that induces HSPs without causing permanent hearing loss. We used this protocol in conjunction with a newly developed mouse model of cisplatin ototoxicity and found that preconditioning mouse inner ears with sound has a robust protective effect against cisplatin-induced hearing loss and hair cell death. Sound therapy also provided protection against aminoglycoside-induced hearing loss. These data indicate that sound preconditioning protects against both classes of ototoxic drugs, and they suggest that sound therapy holds promise for preventing hearing loss in patients receiving these drugs.

Many studies that aim to investigate the underlying mechanisms of hearing loss or balance disorders focus on the hair cells and spiral ganglion neurons of the inner ear. Fewer studies have examined the supporting cells that contact both of these cell types in the cochlea and vestibular end organs. While the roles of supporting cells are still being elucidated, emerging evidence indicates that they serve many functions vital to maintaining healthy populations of hair cells and spiral ganglion neurons. Here we review recent studies that highlight the critical roles supporting cells play in the development, function, survival, death, phagocytosis, and regeneration of other cell types within the inner ear. Many of these roles have also been described for glial cells in other parts of the nervous system, and lessons from these other systems continue to inform our understanding of supporting cell functions. This article is part of a Special Issue entitled “Annual Reviews 2013” .

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear. Since there is no cell line that satisfactorily represents mammalian hair cells, research on hair cells relies on primary organ cultures. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice (Figure 1). The utricle is a vestibular organ, and the hair cells of the utricle are similar in both structure and function to the hair cells in the auditory organ, the organ of Corti. The adult mouse utricle preparation represents a mature sensory epithelium for studies of the molecular signals that regulate the survival, homeostasis, and death of these cells. Mammalian cochlear hair cells are terminally differentiated and are not regenerated when they are lost. In non-mammalian vertebrates, auditory or vestibular hair cell death is followed by robust regeneration which restores hearing and balance functions. Hair cell regeneration is mediated by glia-like supporting cells, which contact the basolateral surfaces of hair cells in the sensory epithelium. Supporting cells are also important mediators of hair cell survival and death. We have recently developed a technique for infection of supporting cells in cultured utricles using adenovirus. Using adenovirus type 5 (dE1/E3) to deliver a transgene containing GFP under the control of the CMV promoter, we find that adenovirus specifically and efficiently infects supporting cells. Supporting cell infection efficiency is approximately 25-50%, and hair cells are not infected (Figure 2). Importantly, we find that adenoviral infection of supporting cells does not result in toxicity to hair cells or supporting cells, as cell counts in Ad-GFP infected utricles are equivalent to those in non-infected utricles (Figure 3). Thus adenovirus-mediated gene expression in supporting cells of cultured utricles provides a powerful tool to study the roles of supporting cells as mediators of hair cell survival, death, and regeneration.

The inner ear transduces sound energy and head motion into neural impulses. These signals are detected by six sensory patches in the fluid-filled spaces of the inner ear. The snail-shaped cochlea detects sound, whereas the vestibular system serves balance and gravity-detection functions. All six sensory patches in the inner ear use mechanosensory hair cells to transduce fluid motion signals into neurotransmitter release. These sensory cells are sensitive to death from noise trauma, aging, and certain therapeutic drugs. Hair cells in nonmammalian vertebrates are regenerated after they die, resulting in functional recovery of hearing and balance. In contrast, mammalian sensory hair cells are not regenerated, and their loss results in permanent hearing and/or balance disorders. Cochlear hair cells make synaptic connections with spiral ganglion neurons. Spiral ganglion neurons are bipolar cells with dendrites that synapse with the basal surfaces of hair cells and axons that comprise the eighth cranial nerve. Hair cells provide trophic support to spiral ganglion neurons. Therefore, death of hair cells is often followed by spiral ganglion neuron degeneration. Hearing loss is the most common sensory impairment in humans and is the sixth most common chronic health problem in the United States. This chapter addresses apoptotic death of sensory hair cells in response to ototoxic drugs and the subsequent death of spiral ganglion neurons (SGNs).

Sensory hair cells of the inner ear are sensitive to death from aging, noise trauma, and ototoxic drugs. Ototoxic drugs include the aminoglycoside antibiotics and the antineoplastic agent cisplatin. Exposure to aminoglycosides results in hair cell death that is mediated by specific apoptotic proteins, including c-Jun N-terminal kinase (JNK) and caspases. Induction of heat shock proteins (Hsps) can inhibit JNK- and caspase-dependent apoptosis in a variety of systems. We have previously shown that heat shock results in robust upregulation of Hsps in the hair cells of the adult mouse utricle in vitro. In addition, heat shock results in significant inhibition of both cisplatin- and aminoglycoside-induced hair cell death. In this system, Hsp70 is the most strongly induced Hsp, which is upregulated over 250-fold at the level of mRNA 2 h after heat shock. Hsp70 overexpression inhibits aminoglycoside-induced hair cell death in vitro. In this study, we utilized Hsp70-overexpressing mice to determine whether Hsp70 is protective in vivo. Both Hsp70-overexpressing mice and their wild-type littermates were treated with systemic kanamycin (700 mg/kg body weight) twice daily for 14 days. While kanamycin treatment resulted in significant hearing loss and hair cell death in wild-type mice, Hsp70-overexpressing mice were significantly protected against aminoglycoside-induced hearing loss and hair cell death. These data indicate that Hsp70 is protective against aminoglycoside-induced ototoxicity in vivo.

Xenopus rods and cones secrete into the interphotoreceptor matrix (IPM) a 124-kDa glycoprotein termed interphotoreceptor retinoid-binding protein (IRBP; Hessler et al. [1996] J. Comp. Neurol. 367:329–341). IRBP is confined to the IPM, being too large to diffuse through the zonulae adherentes between adjacent photoreceptor and Muller cells. Despite this physical entrapment within the subretinal space, IRBP is rapidly cleared from the IPM by an unknown mechanism. Immunohistochemistry and immunoelectron microscopy were used to localize IRBP in intact and detached retina-retinal pigment epithelium (RPE) eyecups. The effects of light, dark, and time of day on the compartmentalization of IRBP were characterized by quantitative Western blot analysis and by immunoprecipitation of IRBP labeled in vivo by intraocular injection of [35S]methionine. Immunohistochemistry showed that the apparent intercellular IRBP in both the RPE and the photoreceptors is resistant to saline extraction, in contrast to that in the IPM. In the RPE, IRBP was associated with matrix material within phagosomes and endosomes. The IPM, RPE, and retina contained 75%, 18%, and 7% of the total IRBP in the eye, respectively. The IPM and RPE contain 130 ± 14 pmoles and 34 ± 4 pmoles of IRBP, respectively. The amounts of IRBP in the RPE at middark and midlight were the same. Furthermore, the in vivo uptake of [35S]methionine-labeled IRBP was light independent. Our studies suggest that IRBP is not strictly confined to the subretinal space but rather that significant amounts are present intracellularly, particularly within the RPE, which does not synthesize IRBP. Furthermore, IRBP secreted by the photoreceptors is taken up from the IPM mainly through a light-independent endocytic pathway separate from outer segment phagocytosis. The role of RPE endocytosis should be explored in relation to the function of IRBP. J. Comp. Neurol. 466:331–342, 2003. © 2003 Wiley-Liss, Inc.

Mechanoreceptive hair cells are extremely sensitive to aminoglycoside antibiotics, including neomycin. Hair cell survival was assessed in larval wild-type zebrafish lateral line neuromasts 4 h after initial exposure to a range of neomycin concentrations for 1 h. Each of the lateral line neuromasts was scored in live fish for the presence or absence of hair cells using the fluorescent vital dye DASPEI to selectively label hair cells. All neuromasts were devoid of DASPEI-labeled hair cells 4 h after 500 microM neomycin exposure. Vital DASPEI staining was proportional to the number of hair cells per neuromast identified in fixed larvae using immunocytochemistry for acetylated tubulin and phalloidin labeling. The time course of hair cell regeneration in the lateral line neuromasts was also analyzed following neomycin-induced damage. Regenerated hair cells were first observed using live DASPEI staining 12 and 24 h following neomycin treatment. The potential role of proliferation in regenerating hair cells was analyzed. A 1 h pulse-fix protocol using bromodeoxyuridine (BrdU) incorporation was used to identify S-phase cells in neuromasts. BrdU incorporation in neomycin-damaged neuromasts did not differ from control neuromasts 4 h after drug exposure but was dramatically upregulated after 12 h. The proliferative cells identified during a 1 h period at 12 h after neomycin treatment were able to give rise to new hair cells by 24-48 h after drug treatment. The results presented here provide a standardized preparation for studying and identifying genes that influence vertebrate hair cell death, survival, and regeneration following ototoxic insults.

Aminoglycoside exposure results in the apoptotic destruction of auditory and vestibular hair cells. This ototoxic hair cell death is prevented by broad-spectrum caspase inhibition. We have used in situ substrate detection, immunohistochemistry, and specific caspase inhibitors to determine which caspases are activated in the hair cells of the adult mouse utricle in response to neomycin exposure in vitro. In addition, we have examined the hierarchy of caspase activation. Our data indicate that both upstream caspase-8 and upstream caspase-9, as well as downstream caspase-3 are activated in hair cells exposed to neomycin. The inhibition of caspase-9-like activity provided significant protection of hair cells exposed to neomycin, whereas the inhibition of caspase-8-like activity was not effective in preventing neomycin-induced hair cell death. In addition, caspase-9 inhibition prevented the activation of downstream caspase-3, whereas the inhibition of caspase-8 did not. These data indicate that caspase-9 is the primary upstream caspase mediating neomycin-induced hair cell death in this preparation.

Mutations in over 70 genes have now been identified as causing hereditary hearing loss in humans. This expanding knowledge about the genetics of hearing loss opens the door for the development of gene therapies aimed at restoring hearing function by delivering corrective DNA to the inner ears of patients with hearing loss caused by these mutations. This review summarizes some of the recent advances toward the goal of developing gene therapy as a treatment for hereditary hearing loss in humans.